13, 14 Our recent results further indicate that high-risk hepatoblast-like HCC characterized by the worst prognosis may be resistant to treatment with epigenetic-based treatment modalities.15 Therefore, we hypothesized that epigenetic modulation would not affect core CSCs due to their intrinsic property of multidrug resistance while promoting the differentiated status of non-CSCs, thereby forcing them out of the SP pool.16-18 The specific objectives were to (1) address the utility of epigenetic modulation for prospective isolation of core CSCs using short-term treatment of liver cancer cell lines with the potent

DNA methyltransferase (DNMT)-1 inhibitor zebularine (ZEB), (2) validate the findings in primary human cancers, and www.selleckchem.com/products/VX-809.html (3) provide an in-depth functional and molecular characterization of isolated CSCs using integrative genomics analysis. Our results demonstrate that ZEB treatment increased representation of highly

tumorigenic cells with CSC properties within the SP fraction from cancer cell lines and primary cancer cells while reducing the tumorigenic capacity of the non-SP fraction. Transcriptome analyses of the CSC expression signatures revealed the diversity of activated oncogenic pathways and preservation of the core PS-341 stemness-associated gene-sets. CSC, cancer stem cells; DMEM, Dulbecco’s modified Eagle’s medium; DNMT, DNA methyltransferase; GFP, green fluorescent protein;

GSEA, gene set enrichment analysis; FACS, fluorescence-activated cell sorting; HCC, hepatocellular carcinoma; NF-κB, nuclear factor κB; NOD/SCID, nonobese diabetic/severe combined imunodeficient; MycoClean Mycoplasma Removal Kit SP, side population; ZEB, zebularine. Detailed descriptions of hepatoma cell lines are provided in the Supporting Information. Cells were treated with 100 μM ZEB (Drug Synthesis and Chemistry Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute) for 3 days. Optimal dose (100 μM) was selected based on published data and efficiency in reducing SP fraction without overt cell toxicity. Primary tumors were obtained from patients undergoing surgery at the NCI Surgery Branch and from the UPMC, Pittsburgh in accordance with ethical guidelines. Tumors were processed directly or after one round of xenotransplantation as described in the Supporting Information. SP analysis was performed as described.19 Cells were incubated at 37°C for 90 minutes with 15 μg/mL of Hoechst-33342 (Invitrogen), either alone or in presence of 50 μmol/L of Fumitremorgin C (Sigma). Only viable cells underwent fluorescence-activated cell sorting (FACS) (based on 7-AAD exclusion) followed by confirmation of purity and viability and used only when both parameters were >90%. All procedures were performed with approval of and in accordance with the guidelines of the National Institutes of Health animal care committee.